Liver transplantation currently is the only mode of treatment for patients in acute fulminant hepatic failure who are not responding to supportive therapy (Starzl et al. "Liver Transplantation (1)" N Engl J Med (1989) 321:1092-1099; Langer and Vacanti "Tissue Engineering" Science (1993) 260:920-926). The need for an interim liver assist device as a bridge to transplantation for patients in hepatic failure has been well documented (Takahashi et al. "Artificial Liver: State of the Art" Dig Diseases Sci (1991) 36:1327-1340). With the development of an artificial liver, patients in hepatic failure may be supported until donor livers become available or until their own livers can regenerate. Such a device would alleviate the problem of scarcity of donor organs (Busuttil et al. "The First 100 Liver Transplants at UCLA" Ann Surg (1987) 206:387-402; Vacanti et al. "Liver Transplantation in Children: The Boston Center Experience in the First 30 Months" Transplant Proc (1987) 19:3261-3266.) and associated complications (Walvatne & Cerra "Hepatic Dysfunction in Multiple Organ Failure" In Multiple Organ Failure: Pathophysiology and Basic Concepts of Therapy, Dietch, E. A., Ed., (1990) pp. 241-260, Thieme Medical Publishers, New York; Shellman et al. "Prognosis of Patients with Cirrhosis and Chronic Liver Disease Admitted to the Medical Intensive Care Unit" Crit Care Med (1988) 16:671-678).
Animal cells and genetically altered derivatives thereof often are cultivated in bioreactors for the continuous production of vaccines, monoclonal antibodies and pharmaceutic proteins, such as hormones, antigens, tissue type plasminogen activators and the like. The cells essentially are a system of catalysts and the medium supplies and removes the nutrients and growth inhibiting metabolites. To supply nutrients and remove metabolites, the medium in the bioreactor is changed either intermittently or continuously by fluid flow. However, because of relatively small size and small density difference when compared to the medium, cells inevitably are withdrawn when the medium is changed, resulting in a relatively low cell concentration within the bioreactor. As a result of the low cell concentration, the concentration of the desired cell product is low in the harvested medium.
An ideal animal cell bioreactor would include three features:
(1) cells would be retained in a viable state at high densities in the bioreactor apparatus as long as possible, with an almost infinite residence time; PA1 (2) high molecular weight compounds, including expensive growth factors and the desired cell products, would have a long but finite residence time within the bioreactor to allow for both efficient nutrient utilization by the growing cells and also the accumulation of cell products to a high concentration; and PA1 (3) low molecular weight compounds, including less expensive nutrients and inhibitory substances, should have a very short residence time within the bioreactor to reduce inhibition of cell growth, cell product formation and other cellular metabolic activities.
The development of an artificial liver is a complex problem. Many prior attempts, such as plasmapheresis, charcoal and resin hemoperfusion and xenograft cross circulation, have failed. Unlike the heart that has one major physiologic function, the liver performs many complex tasks necessary for survival. Those tasks have been difficult to develop or maintain in mechanical systems.
The liver is the metabolic factory required for the biotransformation of both endogenous and exogenous waste molecules and the synthesis of glucose, lipids and proteins, including albumin, enzymes, clotting factors and carrier molecules for trace elements. The liver maintains appropriate plasma concentrations of amino and fatty acids as well as detoxifying nitrogenous wastes, drugs and other chemicals. Waste products, such as bilirubin, are conjugated and excreted via the biliary tree. Hepatic protein synthesis and biotransformation vastly increase the complexity of hepatic support.
Systems that employ hepatocytes to provide biochemical function are problematic because hepatocytes can be difficult to maintain in culture. Under standard conditions, non-transformed hepatocytes cultured on plastic lose gap junctions in about 12 to 24 hours; flatten, become agranular, lose all tissue specific functions in 3-5 days; and die within 1-2 weeks. (Reid & Jefferson "Culturing hepatocytes and other differentiated cells" Hepatology (1984) May-June; 4(3): 548-59; Warren et al. "Influence of medium composition on 7-alkoxycoumarin O-dealkylase activities of rat hepatocytes in primary maintenance culture" Zenobiotica (1988) 18(8):973-81).
A solution to that problem is the use of transformed hepatocytes which can be grown much more easily. However, transformed hepatocytes often are considered a poor choice because even well-differentiated transformed cells show marked variations in tissue-specific function from the parent tissues. (Reid & Jefferson (1984) supra) Moreover, many cell lines are transformed by viruses. (Aden et al. "Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line" Nature (1979) pp. 615-6; Knowles et al. "Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen" Science (1980) 209:497-9). Those cell lines have the potential to transmit the transforming virus to the patient. As a result, it is doubtful that regulatory agencies would approve the use of transformed cells for humans, even if the risk of transmission were proven minimal.
Many approaches to prolonging the viability and function of cultured hepatocytes and other differentiated cells have been investigated. Those approaches include adding hormones and growth factors to the culture media, adding extracellular matrix constituents and growing the hepatocytes in the presence of another cell type. Cells routinely used in co-culture work with hepatocytes are endothelial cells or hepatic nonparenchymal cells, such as Kupffer cells.
The addition of corticosteroids to the incubation media has been shown to prolong survival of cultured hepatocytes and to maintain albumin synthesis, particularly in synergy with insulin. (Jefferson et al. "Post-transcriptional modulation of gene expression in cultured rat hepatocytes" Mol Cell Biol (1984) 4(9):1929-34; Dich et al. "Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity" Hepatology (1988) 8(1):39-45) DMSO (Dimethyl sulfoxide) and phenobarbital also are known to prolong hepatocyte viability and function. (Maher, J. J. "Primary hepatocyte culture: is it home away from home?" Hepatology (1988) 8(5):1162-6) Not all tissue-specific functions are supported equally, however. Insulin can promote some functions with an effect that varies with concentration. If only insulin is added to the medium, urea cycle enzyme expression is decreased. That negative effect can be counteracted by the addition of glucagon and dexamethasone. (Dich et al. (1988) supra)
Hormonally-defined medium also can prolong hepatocyte function and viability. (Jefferson et al. (1984) supra) Using a serum-free hormonally-defined medium, good function in baboon hepatocytes has been shown for over 70 days. The medium consisted of epidermal growth factor (100 ng/ml), insulin (10 .mu.g/ml), glucagon (4 mg/ml), albumin (0.5 mg/ml), linoleic acid (5 mg/ml), hydrocortisone (10.sup.-6 M), selenium (10.sup.-7 M), cholera toxin (2 ng/ml), glycyl-histidyl-lysine (20 ng/ml), transferrin (5 mg/ml), ethanolamine prolactin (10.sup.-6 M), (100 ng/ml), somatotropin (1 mg/ml) and thyrotropin releasing factor (10.sup.-6 M). (Lanford et al. "Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium" In Vitro Cell Dev Biol (1989) 25(2):174-82)
It now is clear that the extracellular matrix has considerable influence on cell function and survival. (Bissell & Aggeler "Dynamic reciprocity: How do extracellular matrix and hormones direct gene expression" Mechanisms of Signal Transduction by Hormones and Growth Factors Alan R. Liss, Inc. (1987) 251-62.3) Matrix elements have been shown to reduce or obviate the need for specific growth factors. Using extracted hepatic connective tissue, hepatocytes have been cultured for over 5 months and maintained albumin synthesis for at least 100 days. That extract represented approximately 1% of the liver by weight. One-third of the extract was composed of carbohydrates and noncollagenous proteins; the other two-thirds were collagens, 43% Type I, 43% Type III, and the remainder, an undefined mixture of others including Type IV. (Rojkind et al. "Connective tissue Biomatrix: Its Isolation and Utilization for Long-term Cultures of Normal Rat Hepatocytes" J Cell Biol (1980) 87:255-63) That mixture may not reflect accurately the local hepatocyte environment, the peri-sinusoidal space or Space of Disse.
The presence of matrix in the Space of Disse has been controversial. Some researchers initially suggested that the peri-sinusoidal space was "empty". It now is appreciated that all of the major constituents of basement membrane are present in or around the Space of Disse. (Bissell & Choun "The role of extracellular matrix in normal liver" Scand J Gastroenterol (1988) 23(Suppl 151):1-7)
Heparan sulfate proteoglycan binds both cell growth factors and cells. (Saksela et al. "Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation" J Cell Biol (1988) 107(2):743-51; Gordon et al. "Heparan sulfate is necessary for adhesive interactions between human early hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment" Leukemia (1988) 2(12):804-9) Heparan sulfate may effect directly the hepatocyte nucleus. (Ishihara et al. "Transport of heparan sulfate into the nuclei of hepatocytes" J Biol Chem (1986) 261(29):13575-80), Hepatocytes secrete relatively abundant quantities of heparan sulfate in culture. (Arenson et al. "Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan" Gastroenterology (1988) 95(2):441-7) Immunologic studies identified Type I collagen, Type III collagen, Type IV collagen, fibronectin and laminin in the Space of Disse. (Geerts et al. "Immunogold localization of procollagen III, fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver". Histochemistry (1986) 84(4-6):355-62; Martinez-Hernandez, A. "The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver" Lab Invest (1984) 51(1):57-74) There normally is little Type I collagen in the Space of Disse, although hepatocytes in culture show increasing Type I synthesis with de-differentiation, at the expense of Type III collagen synthesis. That effect is reversed with culture techniques that support tissue-specific hepatocyte activity.
Hepatocytes also can be cultured on Matrigel.TM., a biomatrix produced by a sarcoma cell line (EHS). Matrigel.TM. contains Type IV collagen, laminin, entactin and heparan sulfate. On Matrigel.TM., hepatocytes maintain normal albumin synthesis for 21 days. (Bissell & Aggeler (1987) supra).
Close duplication of the normal environment of the hepatocyte also has been attempted by culturing hepatocytes in a confluent monolayer on collagen. A second layer of Type I collagen is added to recreate the normal matrix "sandwich" formed on the "top" and on the "bottom" of the hepatocyte. That technique has shown significantly improved viability and function with albumin synthesis for more than 42 days. (Dunn et al. "Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration" FASEB (1989) 3:174-7)
The effect of various proteoglycans and glycosaminoglycans on gap junction protein synthesis and genetic expression also has been examined carefully. The most effective compounds were dermatan sulfate proteoglycan, chondroitin sulfate proteoglycan, and heparan. Heparan extracted from the liver was most effective. Lambda carrageenan, a seaweed extract, also was effective. (Spray et al. "Proteoglycans and Glycosaminoglycans Induce Gap Junction Synthesis and Function in Primary Liver Cultures" J Cell Biol (1987) 105:541-55) Finally, chitosan, a polysaccharide found in crustacean shells and fungal membranes, has been suggested as a factor that can mimic normal matrix and promote function and survival of cells. (Muzzarelli et al. "Biological activity of chitosan: ultrastructural study" Biomaterials (1988) 9(3):247-52; Scholz & Hu "A two compartment cell entrapment bioreactor with three different holding times for cells, high and low molecular weight compounds" Cytotechnology (1990) 4:127-137).
Another successful technique for culturing differentiated liver cells involves co-culture with nonparenchymal cells. Recently, co-culture of hepatocytes on various endothelial lines was compared. Co-culture showed significantly improved albumin synthesis and maintenance of gap junctions. The cells were grown in the presence of insulin and dexamethasone. The addition of serum did not improve the results. The improved survival and function conferred by co-culture occurred only with cells in close proximity and was not transferred by cell supernatants. (Goulet et al. "Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes" Hepatology (1988) 8(5):1010-8).
It still is controversial whether the beneficial effects of co-culture occur through matrix interactions or require cell-cell contact.
There also is evidence that lipocytes play a key role in matrix production. Lipocytes are reported to be as numerous as Kupffer cells and have been suggested to produce the majority of Type I collagen, Type II collagen, Type IV collagen, laminin and proteoglycans, particularly dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan. (Friedman et al. "Hepatic lipocytes: The principle collagen-producing cells of normal rat liver" PNAS (1985) 82:8681-5) It is of particular interest that those specific proteoglycans were those that best support gap junctions (Spray et al. (1987) supra).
Many techniques of artificial support have been utilized over the past three and a half decades. Those include simple exchange transfusions (Lee & Tink "Exchange transfusion in hepatic coma: report of a case" The Med J Australia (1958) 11:40-42; Trey et al. "Treatment of hepatic coma by exchange blood transfusion" NEJM (1966) 274(9):473-81); plasmapheresis with plasma exchange (Sabin & Merritt "Treatment of hepatic coma in cirrhosis by plasmapheresis and plasma infusion [plasma exchange]" Annals of Internal Medicine (1968) 68(1):1-6); extracorporeal heterologous or homologous liver perfusion (Eisemann et al. "Heterologous liver perfusion in treatment of hepatic failure" Annals of Surgery (1965) 162(3):329-345; Sen et al. "Use of isolated perfused cadaveric liver in the management of hepatic failure" Surgery (1966) 59(5):774-781); cross-circulation (Burnell et al. "Acute hepatic coma treated by cross-circulation or exchange transfusions" NEJM (1967) 276(17):943-953); hemodialysis (Opolon et al. "Hepatic failure coma (HFC) treated by polyacrylonitrile membrane (PAN) hemodialysis (HD)" Trans ASAIO (1976) 22:701-710); activated charcoal hemoperfusion (Gazzard et al. "Charcoal haemoperfusion in the treatment of fulminant hepatic failure" Lancet i:1301-1307); and, more recently, bioartificial liver systems (BAL's) containing cultured hepatocytes.
Examples of bioartificial liver systems currently being investigated for support of liver failure include extracorporeal bioreactors (Arnaout et al. "Development of bioartificial liver: bilirubin conjugation in Gunn rats" Journal of Surgical Research (1990) 48:379-382; Margulis et al. "Temporary organ substitution by hemoperfusion through suspense of active donor hepatocytes in a total complex of intensive therapy in patients with acute hepatic insufficiency" Resuscitation (1989) 18:85-94); implantable hepatocyte cultures, such as microencapsulated gel droplets (Cai et al. "Microencapsulated hepatocytes for bioartificial liver support" Artificial Organs (1988) 12(5):388-393) and spheroid aggregates (Saito et al. "Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen" Transplantation Proceedings (1989) 21(1) :2374-77).
Those bioartificial liver systems have the advantage of performing detoxification, synthesis and bioprocessing functions of the normal liver. Only a few extracorporeal bioreactors have been used in the clinical setting (Matsumura et al. "Hybrid bioartificial liver in hepatic failure: preliminary clinical report" Surgery (1987) 101(1):99-103; Margulis et al. (1989) supra). Implantable hepatocyte cultures remain clinically untested.
The technique for hepatocyte entrapment within microencapsulated gel droplets (hepatocyte microencapsulation) is similar to the technique successfully used for pancreatic islet encapsulation (O'Shea & Sun "Encapsulation of rat islets of Langerhans prolongs xenograft survival in diabetic mice" Diabetes (1986) 35:943-46; Cai et al. (1988) supra). Microencapsulation allows nutrient diffusion to the hepatocytes and metabolite and synthetic production diffusion from the hepatocytes. Microencapsulation also provides intraperitoneal hepatocytes with "immuno-isolation" from the host defenses (Wong & Chang "The viability and regeneration of artificial cell microencapsulated rat hepatocyte xenograft transplants in mice" Biomat Art Cells Art Org (1988) 16(4):731-739).
Plasma protein and albumin synthesis (Sun et al. "Microencapsulated hepatocytes as a bioartificial liver" Trans ASAIO (1986) 32:39-41; Cai et al. (1988) supra); cytochrome P450 activity and conjugation activity (Tompkins et al. "Enzymatic function of alginate immobilized rate hepatocytes" Biotechnol Bioeng (1988) 31:11-18); gluconeogenesis (Miura et al. "Liver functions in hepatocytes entrapped within calcium alginate" Ann NY Acad Sci (1988) 542:531-32); ureagenesis (Sun et al. "Microencapsulated hepatocytes: an in vitro and in vivo study" Biomat Art Cells Art Org (1987) 15:483-486); and hepatic stimulating substance production (Kashani & Chang "Release of hepatic stimulatory substance from cultures of free and microencapsulated hepatocytes: preliminary report" Biomat Art Cells Art Org (1988) 16(4):741-746) all have been reported for calcium alginate-entrapped hepatocytes.
Aggregated hepatocytes have been proposed as a treatment means for fulminant hepatic failure. Multiple techniques exist for hepatocyte aggregation (Saito et al. "Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen" Transplantation Proceedings (1989) 21(1):2374-77; Koide et al. "Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans" Biochem Biophys Res Comm (1989) 161(1):385-91).
Extracorporeal bioreactor designs for the purpose of artificial liver support have included perfusion of small liver cubes (Lie et al. "Successful treatment of hepatic coma by a new artificial liver device in the pig" Res Exp Med (1985) 185:483-494) dialysis against a hepatocyte suspension (Matsumura et al. (1987) supra; Margulis et al. (1989) supra); perfusion of multiple parallel plates (Uchino et al. "A hybrid bioartificial liver composed of multiplated hepatocyte monolayers" Trans ASAIO (1988) 34:972-977); and hollow fiber perfusion. Human studies using extracorporeal hepatocyte suspensions have been reported.
The first clinical report of artificial liver support by dialysis against a hepatocyte suspension was released in 1987 (Matsumura et al. (1987) supra). The device consisted of a rabbit hepatocyte liquid suspension (1-2 liters) separated from patient blood by a cellulose acetate dialysis membrane. Each treatment used fresh hepatocytes during a single four to six hour dialysis (run). Multiple runs successfully reduced serum bilirubin and reversed metabolic encephalopathy in a single case.
A controlled study from the USSR comparing dialysis against a hepatocyte suspension with standard medical therapy for support of acute liver failure was reported recently (Margulis et al. (1989) supra). The bioartificial device consisted of a small 20 ml cartridge filled with pig hepatocytes in liquid suspension, along with activated charcoal granules. The cartridge was perfused through a Scribner arteriovenous shunt access. Patients were treated daily for six hours. The hepatocyte suspension was changed hourly over each six hour treatment period. Improved survival was demonstrated in the treated group (63%) when compared with the standard medical therapy control group (41%).
Culturing hepatocytes with a hollow fiber cartridge is another example of bioartificial liver support. Traditionally, hepatocytes are loaded in the extracapillary space of the hollow fiber cartridge, while medium, blood or plasma is perfused through the lumen of the hollow fibers. Cells may be free in suspension (Wolf & Munkelt "Bilirubin conjugation by an artificial liver composed of cultured cells and synthetic capillaries" Trans ASAIO (1975) 21:16-27); attached to walls (Hager et al. "A Prototype For A Hybrid Artificial Liver" Trans ASAIO (1978) 24:250-253); or attached to microcarriers which significantly increase the surface area within the extracapillary space (Arnaout et al. (1990) supra).
Bilirubin uptake, conjugation and excretion by Reuber hepatoma cells within a hollow fiber cartridge was reported in 1975. (Wolf & Munkelt (1975) supra). Tumor cell suspensions were injected by syringe into the shell side of the compartment while bilirubin containing medium was perfused through the hollow fiber intraluminal space. That technique has not been reported clinically, possibly due to the risk of tumor seeding by hepatoma cells.
Another hollow fiber device developed for liver support uses hepatocytes attached to microcarriers loaded into the extracapillary cavity of a hollow fiber cartridge. In that device, blood flows through semi-permeable hollow fibers allowing the exchange of small molecules. Using that system, increased conjugated bilirubin levels have been measured in the bile of glucuronosyl transferase deficient (Gunn) rats. (Arnaout et al. "Development of Bioartificial Liver: Bilirubin Conjugation in Gunn Rats" J Surg Research (1990) 48:379-82) Since the outer shell is not perfused, all oxygen and nutrients are provided by the patient blood stream. In addition, that system may require an intact in vivo biliary tree for the excretion of biliary and toxic wastes.
However, for clinical applications, it is desirable to increase the liver-specific functions of the BAL, thereby requiring more cells or increasing the per cell liver-specific function. The former avenue generally is not considered because normal cells are difficult to obtain, the cells are difficult to maintain and the bioreactor cannot command a large blood volume during ex vivo therapy.
Primary rat hepatocytes, when plated on some modified surfaces, form aggregates that exhibit enhanced per cell liver-specific functions (Koide et al. "Continued High Albumin Production by Multicellular Spheroids of Adult Rat Hepatocytes Formed in the Presence of Liver-Derived Proteoglycans" Biochem Biophys Res Commun (1989) 161:385-391; Tong et al. "Long-Term Culture of Adult Rat Hepatocyte Spheroids" Exp Cell Res (1992) 200:326-332). Freshly isolated rat hepatocytes, when plated between 30-80% confluency onto positively charged polystyrene surfaces (Koide et al. "Formation of Multicellular Spheroids Composed of Adult Rat Hepatocytes in Dishes with Positively Charged Surfaces and Under Other Nonadherent Environments" Exp Cell Res (1990) 186:227-35), initially spread out and seem to move randomly. After 48 hours, cell movement appears directional as cells begin to aggregate into multicellular islands which eventually shed off into suspension as freely suspended aggregates. Aggregates formed in that manner exhibit a uniform diameter of approximately 100 .mu.m and are 6-8 cell layers thick.
Reported systems for making aggregates include culture of aggregates in a polyurethane foam matrix in a packed bed culture system (Ijima et al. "Application of Three Dimensional Culture of Adult Rat Hepatocytes in PUF Pores for Artificial Liver Support System" In: Animal Cell Technology: Basic & Applied Aspects Murakami et al. Ed., (1992) pp 81-86, Kluwer Academic Publishers, The Hague, Netherlands), culture of aggregates in a tubular reactor packed with pyrex glass beads (Li et al. "Culturing of Primary Hepatocytes as Entrapped Aggregates in a Packed Bed Bioreactor: A Potential Bioartificial Liver" In Vitro Cell Dev Biol (1993) 29A:249-254), culture of calcium alginate-encapsulated aggregates in a spouted bed culture chamber (Takabatake et al. "Encapsulated Multicellular Spheroids of Rat Hepatocytes Produce Albumin and Urea in a Spouted Bed Circulating Culture System" Artif Organs (1991) 15:474-480; Koide et al. "Hepatocyte Spheroid: Differentiated Features and Potential Utilization for Bioreactor of Artificial Liver Support" Extended Abstract, Japanese Association of Animal Cell Technology Annual Meeting, Nov. 9-12, 1993, Nagoya, Japan) and collagen-entrapped aggregates inoculated into the extracapillary space of a hollow fiber bioreactor (Sakai and Suzuki "A Hollow Fiber Type Bioartificial Liver Using Hepatocyte Spheroids Entrapped in Collagen Gel" Extended Abstract, Japanese Association of Animal Cell Technology Annual Meeting, 1993, Nagoya, Japan).
A common limitation of each of those systems is the low number of hepatocytes attainable for use in the bioreactor. Approximately 50 million through 75 million hepatocytes as aggregates were used in those studies. The aggregate formation process using stationary petri dishes or other surfaces is long and labor intensive. Aggregate formation occurs only within a narrow cell density range (approximately 3-8.times.10.sup.4 cells/cm.sup.2). Of the cells initially plated, only 30-40% of the inoculated cells form aggregates after 2-3 days of culture. Thus, to supply 100 million hepatocytes as aggregates, an inoculum of approximately 300-400 million cells is required. Based on plating density requirements, that translates to a surface area of 8000 cm.sup.2 or 200 petri dishes of 60 mm diameter. Thus, the feasibility of employing reconstituted hepatocytes (aggregates) in a bioartificial liver application depends on the ability to engineer reconstituted hepatocyte formation at a quicker rate and a higher efficiency.
The availability of a higher number of aggregates would enable maximization of viable cells in the BAL without detrimentally increasing the size of the device.